Functional characterization of LFA-1 antigens in the interaction of human NK clones and target cells

In the present studies we analyzed the role of LFA-1 antigens in the interaction between NK clones and target cells. The use of various cloned NK cell lines allowed us to analyze homogeneous populations of NK cells which ordinarily comprise only a small fraction of peripheral blood lymphocytes and are extremely heterogeneous with respect to phenotype and specificity. Indirect immunofluorescence with monoclonal antibodies against the alpha (MHM24) and beta (MHM23) chains of the LFA-1 antigen revealed similar patterns of positive reactivity with all NK clones. Both monoclonal antibodies exerted a significant blocking effect on NK cytotoxicity against target cells such as Molt-4 and CEM, whereas the inhibition was very weak against other targets such as K562 and HSB cells. Additive blocking effects were seen when both monoclonal antibodies MHM23 and MHM24 were added to the cytotoxicity assays. When we compared the inhibitory effect of MHM23 and MHM24 on uncultured peripheral blood NK cells and IL 2-activated NK cells, inhibition of cytotoxicity also was found to be primarily dependent on the individual target cells. Thus, the inhibitory activity of anti-LFA-1 antibody was shown to be independent of the phenotypic and functional heterogeneity of the NK clones, activated NK cells, and unstimulated NK cells utilized in these studies. These blocking effects were found to be independent of the LFA-1 antigen expression on the target cell membrane and inhibition occurred only when antibody was bound to the effector cells. Comparison of the effects of anti-LFA-1, anti-T3, and anti-clonotypic antibodies against a Ti-like structure of different NK clones with a mature T cell phenotype demonstrated that each of these antibodies acts on the effector cells in an independent and additive fashion. However, unlike T3 and NKTa antigen, LFA-1 antigen expression is not modulated by cell surface interaction with antibodies specific for this molecule.     

Antigen-specific human T lymphocyte clones: viral antigen specificity of influenza virus-immune clones

Human T lymphocyte clones (TLC) specific for type A (A/Texas/1/77) influenza virus and maintained in continuous culture with T cell growth factor, were analyzed to define the cellular specificity pattern of virus recognition. A panel of TLC were stimulated with strains of serologically characterized type A influenza subtypes. Five TLC recognized all the viral subtypes; the remaining clones recognized only subtypes that shared serologically defined determinants with the immunizing subtype. In addition, the 11 TLC were analyzed for their fine antigenic specificity by using the purified viral components hemagglutinin (HA), neuraminidase (NA), matrix protein (MP), and nucleoprotein (NP). Five TLC proliferated in response to NA, four to MP, one to HA, and one to NP. None of the clones responded to the unrelated B strain influenza virus, B/Singapore. Furthermore, the fine specificity of an MP-reactive TLC was confirmed by subcloning.     

Alloreactive bovine T lymphocyte clones: an analysis of function, phenotype, and specificity

A bovine alloreactive cell population was subjected to complement-dependent lysis with monoclonal antibody (mAb) IL-A11. The original population and the population depleted of cells bearing the determinant recognized by mAb IL-A11 were cloned. Parent cultures and 21 clones were examined for cytolytic function and for expression of determinants recognized by mAb IL-A11 and two additional mAb, IL-A12 and IL-A17. Clones could be classified according to maximal achievable levels of cytolysis by using Theileria parva-infected bovine lymphoblastoid target cells. In this way, three groups were identified--one capable of high level cytolysis, one of intermediate levels, and one group comprising apparently noncytolytic clones. The clones in the first group reacted with mAb IL-A17; those in the second and third groups, with mAb IL-A11 and IL-A12. It was shown that cytotoxicity effected by IL-A17+ clones could be inhibited by this mAb and also by a mAb directed to MHC class I determinants on target cells. Conversely, cytotoxicity effected by IL-A11+/IL-A12+ clones could be inhibited by mAb IL-A11 and by a mAb directed to MHC class II determinants on target cells. The levels of expression of class I and class II determinants on target cells correlated with the levels of killing by clones of the IL-A17+ phenotype and clones of the IL-A11+/IL-A12+ phenotype, respectively. The results indicate that cytotoxic bovine T lymphocyte clones specific for class I MHC antigens and both cytotoxic and noncytotoxic clones specific for class II MHC antigens can be obtained. Further, their specificity for class I or class II antigens can be determined by phenotyping with mAb.     

Antigen-specific human T lymphocyte clones: induction, antigen specificity, and MHC restriction of influenza virus-immune clones

Human peripheral blood lymphocytes from an HLA-Dw1,3 individual were primed in vitro with influenza A virus (A/Texas/1-77/x-49) and subsequently cloned by limiting dilution in TCGF. Of the 96 TLCs originally obtained, nine were characterized in detail. TLCs were antigen specific, responding to influenza A virus, not to influenza B, TGAL, GAT, tetanus toxoid, or KLH, and only when antigen was presented by cells unable to form rosettes with AET-treated SRBC. Presentation of antigen by unseparated PBL often resulted in significant "back stimulation," probably via production of growth factors. The MHC requirements for the induction of TLC proliferation were analyzed. Of four representative clones analyzed, three required Dw1;DR1 compatibility for successful presentation of viral antigens by a panel of antigen-presenting cells. In contrast, one TLC showed an unusual pattern of response that could not be correlated to a particular HLA haplotype. Monoclonal anti-T cell antibody analysis of the surface phenotype of two TLCs maintained in continuous culture for 5 mo indicated that they were OKT3+, 4+, and 8-, consistent with an inducer/helper phenotype. To confirm the clonal nature of TLCs, data on the functional properties of TLC subclones are also presented.     

Generation of monoclonal antibodies blocking cytotoxic reactions by human NK clones: further characterization of a 40/80-kDa target cell receptor

To study NK target antigen(s), mice were immunized with pooled cells from five human hematopoietic cell lines (K562, MOLT4, JM, CEM, U937) known to be susceptible to Natural Killer activity. Cells fusions were performed and 4 out of approximately 2000 hybridoma supernatants were selected because of their ability to block cytotoxic reactions between a human NK clone (termed JT9) and MOLT4 cells. Functional characterization of the four monoclonal antibodies (Mabs) indicated that individual treatment of each immunizing target cells resulted in a decreased cytotoxicity. This inhibition was very specific because it was exclusively observed when three clones, JT9, JT10, and JT11, which express the same clonotypic structure (NKTa), were used as effector cells. Parallel and sequential immunoprecipitations showed that the four reagents termed anti-TNKtar 1, 2, 3, and 4 were directed at the same 40/80-kDa heterodimeric structure previously identified by anti-TNKtar Mab. However, cross-blocking experiments indicated that TNKtar and TNKtar 1-4 represent two distinct epitopic clusters. Finally, it was shown that anti-TNKtar 1-4 recognition sites are either identical or closely related to that of an additional antibody termed 4F2. Together, the present data strengthen the hypothesis that the activation antigen recognized by these series of Mab serves as a target cell receptor for at least a minor population of NK active lymphocytes.     

A human NK cell activation/inhibition threshold allows small changes in the target cell surface phenotype to dramatically alter susceptibility to NK cells

NK cell activation is negatively regulated by the expression of target cell MHC class I molecules. We show that this relationship is nonlinear due to an NK cell activation/inhibition threshold. Ewing's sarcoma family tumor cell monolayers, which were highly susceptible to NK cells in vitro, developed a highly resistant phenotype when cultured as three-dimensional multicellular tumor spheroid structures. This suggested that tumor architecture is likely to influence the susceptibility to NK cells in vivo. Resistance of the multicellular tumor spheroid was associated with the increased expression of MHC class I molecules and greatly reduced NK cell activation, implying that a threshold of NK cell activation/inhibition had been crossed. Reducing MHC class I expression on Ewing's sarcoma family tumor monolayers did not alter their susceptibility to NK cells, whereas increased expression of MHC class I rendered them resistant and allowed the threshold point to be identified. This threshold, as defined by MHC class I expression, was predictive of the number of NK-resistant target cells within a population. A threshold permits modest changes in the target cell surface phenotype to profoundly alter the susceptibility to NK cells. Whereas this allows for the efficient detection of target cells, it also provides a route for pathogens and tumors to evade NK cell attack.     

Cellular immune response to human sarcomas: cytotoxic T cell clones reactive with autologous sarcomas. I. Development, phenotype, and specificity

Human T cell clones cytotoxic for autologous sarcoma cell lines have been developed from patient JM with an osteogenic sarcoma, and from patients EG and RM with malignant fibrohistiocytoma. These clones were derived from the cocultivation of peripheral blood lymphocytes (PBL) with the respective patient's autologous irradiated established tumor cell lines (AIT). After two cycles of stimulation for 5 days in bulk culture, these "educated" lymphocytes were seeded at a density of 1 X 10(6) cells/well in 24-well plates and were cultured in the presence of highly purified natural IL 2 and AIT, the latter serving as a feeder layer. Cell numbers were reduced from the initial seeding density by one log each week until reaching a density of 10(2) cells. These cells were found to be stable in viability and cytotoxic activity, after which limiting dilution was then performed. Within 4 to 6 wk, clones were isolated with unique specificities. These clones were capable of proliferating to a total density of 10(9) cells/ml and maintained their specific cytotoxicity for more than 6 mo. Testing with a panel of target cells of various histotypes, cold-target inhibition assays, and blocking of cytotoxicity with anti-HLA monoclonal antibodies showed that the T cell clones recognize a common sarcoma-associated antigen and that the lysis is HLA restricted. Phenotypically, cytotoxic clones derived from JM were Leu-1+, Leu-2+, and Leu-3-, whereas those derived from EG exhibited either Leu-24 or Leu-3+ markers, the latter phenotype lacking cytotoxicity. RM exhibited mainly Leu-3+ clones with strong cytotoxicity. All were HNK-1- and HLA class II+, with less than 1% of cells of each clone stained by anti-TAC monoclonal antibody. The clones from each patient did not lyse autologous or allogeneic PBL, mitogen-induced T lymphoblasts, normal fibroblasts, cells isolated from benign neoplasms, carcinoma cells, Daudi B lymphoid cells, or K562 cells. With the exception of EG, all clones produced immune interferon in a range from 12 to 50 U/ml. The generation of long-term specific T cell clones can be used to further dissect the cellular immune response to sarcomas. Cytotoxic T cell clones have potential application for tumor immunotherapy.     

Hepatitis B virus-reactive human T lymphocyte clones: antigen specificity and helper function for antibody synthesis

To study immunity to hepatitis B surface antigen (HBsAg) at the cellular level, lymphocytes were obtained from the peripheral blood of hepatitis B vaccine recipients and were examined for various immune responses to HBsAg in vitro. The peripheral blood mononuclear cells (PBM) from most of the vaccinees did not proliferate to a great extent to HBsAg in vitro. However, HBsAg-reactive lymphocyte lines and clones were obtained from some of these individuals if the PBM were stimulated in vitro with HBsAg and were maintained in the presence of T cell growth supplement. Most of the HBsAg-reactive T cell clones obtained were found to be antigen-specific and some of them provided help in the production of anti-HBsAg antibodies by a cell population enriched for HBsAg-binding cells. These results indicate that HBsAg-specific T and B cells exist in the circulation of hepatitis B vaccine recipients, although they are at limiting concentrations for the in vitro cell proliferation and antibody production assays.     

Genetic restriction and fine specificity of human T cell clones reactive with rabies virus

Rabies virus-specific T cell clones isolated from a human vaccine recipient were studied for their fine specificity and genetic restriction using synthetic peptides of the viral Ag and mouse fibroblasts transfected with human MHC genes. Two clones were found to react with an epitope present in the rabies glycoprotein, which was presented by the HLA-DR7 molecule. Other T cell clones recognized synthetic epitopes corresponding to the rabies nucleoprotein in association with the HLA-DR7 or HLA-DQw3 molecule, and one clone responded to the viral nucleocapsid Ag in the presence of HLA-DPw4. T cell clones that exhibited different cross-reactivity patterns among several virus strains were found to recognize closely situated epitopes (within 15 amino acid residues), which were presented in the context of the same MHC molecule. The lack of recognition of a particular virus strain by a T cell clone was attributable in some cases to amino acid variations of the Ag that appear to affect the T cell's receptor for Ag specificity and not the ability of that epitope to associate with the corresponding MHC molecule. Comparisons of the T cell cross-reactivity patterns with various rabies and rabies-related viruses, the fine antigenic specificity, and MHC restriction may aid in understanding the role of individual amino acid variations among virus strains in the induction of cross-protective immunity.     

Detection of double target binders at the single cell level: an evaluation of the specificity of NK and MLC-induced NK-like cells

MLC-generated cells were tested on 7 consecutive days in the single cell cytotoxicity assay to determine the kinetics of natural and allospecific killing. Maximum cytotoxicity to the NK-sensitive target, K562, was found on Day 3 of MLC with an increase at that time in both the number of cells binding and the number of cells killing K562. The maximum allospecific response was found on Days 6 and 7 with an increase in cells able to bind and kill the alloantigen-bearing target. To determine whether the anti-K562 and allospecific killing were mediated by the same effector cells or different cell populations, both targets were tested simultaneously in the single cell assay. At no time during the 7 days were cells detected capable of simultaneously binding both K562 and allospecific targets. These data indicate that there are two different cell populations responsible for allospecific cytotoxicity and MLC-induced NK-like cytotoxicity. The cytotoxic specificity of unstimulated and MLC-generated NK-like cells was also investigated. When two different NK-sensitive targets (e.g., K562 and MOLT-4) were tested together in the single cell assay, there was no concurrent binding of targets by either fresh PBL prior to MLC stimulation or Day 3 MLC-generated cells. When unstimulated effector cells were enriched for NK activity by Percoll density gradient centrifugation, only a small number of effector cells simultaneously binding two different NK-sensitive targets was detected in the single cell assay. These results imply that the NK cell population is heterogeneous and composed of subpopulations recognizing diverse target specificities.     

Expression of glycosylphosphatidylinositol-anchored CD59 on target cells enhances human NK cell-mediated cytotoxicity

NK cell-mediated cytotoxicity of target cells is the result of a balance between the activating and inhibitory signals provided by their respective ligand-receptor interactions. In our current study, we have investigated the significance of CD59 on human target cells in modulating this process. A range of CD59 site-specific Abs were used in NK cytotoxicity blocking studies against the CD59-expressing K562 target cell line. Significantly reduced cytotoxicity was observed in the presence of Abs previously shown to lack blocking capacity for C-mediated lysis. We investigated the consequences for alternative membrane attachment modalities, namely bis-myristoylated-peptidyl (BiMP) and GPI anchoring, on CD59-negative U937 cells. Expression of GPI-anchored CD59 either via transfection or incorporation rendered U937 targets more susceptible to NK cytotoxicity, whereas incorporation of CD59 via a BiMP anchor to similar levels did not alter susceptibility to NK cytotoxicity. Localization of both BiMP- and GPI-anchored CD59 proteins was shown to be within the lipid raft microdomain. A role for the GPI anchor and independence from glycosylation status was confirmed by expression of transmembrane-anchored CD59 or unglycosylated CD59 and by testing in NK cytotoxicity assays. To investigate mechanisms, we compared the signaling capacity of the various forms of expressed and incorporated CD59 following Ab cross-linking in calcium flux assays. GPI-anchored CD59, with or without glycosylation, mediated activation events, whereas CD59 forms lacking the GPI anchor did not. The data show that the increased susceptibility of target cells expressing CD59 to NK cytotoxicity requires GPI anchor-mediating signaling events, likely mediated by interactions between GPI-anchored CD59 on targets and NK receptors.     

Isolation and characterization of human T cell lines and clones reactive to rabies virus: antigen specificity and production of interferon-gamma

By using a preparation of inactivated rabies virus, the blood mononuclear cells from five rabies vaccine recipients were stimulated in vitro in the presence of interleukin 2. T cell lines that displayed significant proliferative responses to whole rabies virus and to preparations of rabies glycoprotein and nucleocapsid were obtained from all the individuals. Other antigens, such as diphtheria and tetanus toxoids, influenza A virus, hepatitis B surface antigen, and serum albumin, failed to induce the proliferation of the T cell lines. One of these rabies-specific T cell lines was found to proliferate in response to rabies antigens only when the antigen-presenting cells expressed homologous HLA-DR antigens. The use of mouse monoclonal antibodies specific for human T cell surface markers revealed that most of the cells of these rabies-reactive lines were of the helper/inducer class of T lymphocytes. Stimulation of the T cell lines with the rabies antigens induced the production of interferon-gamma, a lymphokine with potent antiviral activity. Several T cell clones were isolated from two of these cell lines, and most of them appeared to be specific for the antigenic components of the viral nucleocapsid. Two T cell clones specific for the rabies glycoprotein were also isolated from one of these lymphocyte interleukin 2-dependent lines. Further in vitro studies with rabies-specific T cells could help us to understand in more depth the role of regulatory T cells in the human immune response to rabies virus.     

Influenza virus-specific human cytotoxic T cell clones: Heterogeneity in antigenic specificity and restriction by class II MHC products

Human cytotoxic T lymphocytes specific for A/JAP/57 (H2N2) influenza virus were cloned from in vitro stimulations of peripheral blood lymphocytes. Analysis of the viral specificity in cytotoxic function revealed one clone that killed all type A influenza-infected targets, another clone that was specific for the hemagglutinin subtype of the immunizing influenza virus, and the third clone that demonstrated cytotoxicity restricted to the hemagglutinin of A/JAP/57 and A/JAP/62 (H2N2) and not other type A influenza strains with the H2N2 subtypes. The phenotype of these three clones was Leu 2^?, Leu 3⁺, Leu 4⁺; MHC restriction of their cytotoxic function was mapped to HLA-DR by a panel of target cells as well as by inhibition of cytotoxicity with monoclonal antibodies. Proliferation of these clones, examined in a tritiated thymidine incorporation assay, was found to be driven by antigen in the absence of exogenous lymphokines. For all three clones antigen-dependent production and secretion of lymphokines with IL-2 activity was demonstrated. The antigen specificity of proliferation and factor production was shown to be identical to the pattern that each clone revealed in its cytotoxic function.     

The role of interleukin 2 and T11 E rosette antigen in activation and proliferation of human NK clones

Although considerable data have recently been accumulated regarding the functional role of natural killer (NK) cells, relatively little is known about the factors that regulate NK cell activity. In these studies, we evaluated the role of interleukin 2 (IL 2) and the expression of the IL 2 receptor in the activation and proliferation of human NK cloned cell lines. By using a series of cloned cell lines, we were able to analyze homogeneous populations of NK cells that ordinarily comprise only a small fraction of peripheral blood lymphocytes and are extremely heterogeneous with respect to phenotypes and cytotoxic specificities. In comparison with several T cell clones, we found a much lower density of IL 2 receptors on NK clones, regardless of whether or not these cloned cells had a mature T cell phenotype. Correspondingly, NK clones needed a 10-fold higher concentration of recombinant IL 2 for maximal proliferation. Moreover, blocking studies with specific monoclonal IL 2 receptor antibodies indicated that IL 2 is both necessary and sufficient to induce the proliferation of NK clones. Because the majority of peripheral blood NK cells and NK clones express the T11 E rosette receptor antigen, which has been shown to be an antigen-independent activation pathway for T cells, we were able to study the role of monoclonal anti-T11 antibodies in the activation of various NK clones for which a specific target antigen is not known. In contrast to T cell clones, the induction of IL 2 receptor expression after T11 activation was possible only for some NK clones such as JT10 and JT3, but not for CNK5. Before activation, the IL 2 receptor expression of NK clones was confined to cells in the G2 - M phase, but after T11 activation the more pronounced IL 2 receptor expression became independent of the cell cycle. With respect to the direct proliferative effect of anti-T11 activation that has been noted with T cell clones, only the T3+ (JT10) and not the T3- NK clones could be directly stimulated. Nevertheless, IL 2 receptor expression could be triggered on some T3- clones such as JT3. Because T11-induced proliferation of T cells has been shown to be dependent on both the expression of the IL 2 receptor and on the interaction of this receptor with IL 2, it is proposed that the different responses of NK cells to T11 activation may reflect the ability of the individual clone to produce endogenous IL 2, as well as its ability to express the IL 2 receptor.     

Measles virus-specific murine T cell clones: characterization of fine specificity and function

Measles virus (MV)-specific murine helper T cell clones (Thy-1.2+, CD4+, CD8-) were generated from mice immunized with MV-infected mouse brain homogenate by limiting dilution and in vitro stimulation of spleen cells with UV-inactivated MV Ag. The protein specificity of 7 out of 37 stable T cell clones, which displayed MHC-restricted MV Ag recognition, could be assessed by using purified MV proteins. Two fusion (F) protein-specific, two hemagglutinin-specific, and three nucleoprotein- or matrix protein-specific clones were shown to be established. The F protein-specific T cell clones together with a panel of previously generated F protein-specific T cell clones were characterized for their fine specificity by using beta-galactosidase fusion products, which contained different parts of the F protein. It was shown that at least two epitopes on the major part of the F protein (amino acid 2-513) can be recognized by mouse T cells. Functional characterization of three T cell clones showed that they were able to assist MV-specific B cells and bystander B cells for antibody production. Furthermore, they were shown to produce the lymphokines IL-2 and IFN-gamma. It was also shown that these T cell clones induced a MV-specific delayed type hypersensitivity response. These observations suggest that all of the T cell clones characterized belong to the TH1 helper subset.     

Expression of human CD1d molecules protects target cells from NK cell-mediated cytolysis

The cytotoxic activity of NK cells can be inhibited by classical and nonclassical MHC molecules. The CD1 system is formed by a family of glycoproteins that are related to classical MHC. CD1a, b, and c molecules present lipids or glycolipids to T cells and are involved in defense against microbial infections, especially mycobacteria. It has been shown recently that these molecules can inhibit target cell lysis by human NK cells. It has also been shown that mouse CD1d molecules can protect cells from NK cell-mediated cytotoxicity. In the present study, we describe how human CD1d, orthologous to murine CD1 molecules, can inhibit NK cell-mediated cytolysis. We have expressed CD1d in the HLA class I-deficient cell lines L721.221 and C1R. The inhibitory effect is observed when effector NK cells from different donors are used, as well as in different cell lines with NK activity. The inhibitory effect was reversed by incubating the target cells with a mAb specific for human CD1d. Incubation of target cells with the ligands for CD1d, alpha-galactosylceramide (alpha-GalCer), and beta-GalCer abolishes the protective effect of CD1d in our in vitro killing assays. Staining the effector cells using CD1d tetramers loaded with alpha-GalCer was negative, suggesting that the putative inhibitory receptor does not recognize CD1d molecules loaded with alpha-GalCer.     

Characterization of three different rat T-cell clones with specificity to Listeria monocytogenes: phenotype, specific proliferation, lymphokine production, and protective capacity in vivo

Splenic T lymphocytes from rats immunized with the facultative intracellular bacterium, Listeria monocytogenes, were cloned by the limiting-dilution technique. From several clones obtained, three have been scrutinized in detail. As demonstrated by their reactivity to the monoclonal antibodies, W3/25 and MRC OX8, the clones RVIIC2 and R23D6 are of helper cell phenotype, whereas cells from clone R30D5 express both the helper and the cytotoxic/suppressor cell markers. Proliferation of all three clones critically depends on antigen-presenting cells, exogenous interleukin 2, Listeria antigen, and on class II-restricted antigen presentation by accessory cells. There are differences between cells from different clones with respect to the degree of production of migration inhibitory and macrophage-activating factors. Thus, T lymphocytes of clones R23D6 and R30D5 are highly active, whereas cells of clone RVIIC2 showed markedly less production of these factors. In vivo studies, analyzing the capacity of cells to transfer systemic protection, showed a positive correlation between the production of migration inhibitory factor, macrophage-activating factor, and systemic protection.     

Characteristics of seven clones of the WiDr human colon adenocarcinoma cell line

Summary Seven clonal populations were derived from the WiDr human colon adenocarcinoma cell line and were characterized with respect to chromosome number, DNA content, and tumorigenicity in nude mide. There was no correlation between tumor volume and either DNA content or chromosome number; but there were wide differences among the clones regarding the size of tumors they were able to produce in nude mice.